Abstract A vaccine for prevention of genital herpes is a high public health priority. Herpes vaccines that performed well in rodents have not protected humans; therefore, novel approaches are needed. We propose the following: 1) Set a much higher bar for vaccine efficacy in animal models than used previously. The ?gold standard? for a herpes vaccine is sterilizing immunity, defined as no genital lesions and no evidence of subclinical infection as measured by negative day 2 vaginal titers post-infection, no recurrent episodes of vaginal shedding of HSV-2 DNA and no HSV-2 DNA in dorsal root ganglia. Prior herpes candidate vaccines have prevented death and reduced genital lesions, but none has come close to achieving sterilizing immunity by preventing subclinical infection. We have set sterilizing immunity in >95% of animals as our goal. 2) Devise strategies to prevent the virus from evading vaccine-induced immunity. Antibodies correlate with protection for the vast majority of our effective human vaccines. HSV encodes glycoproteins C (gC) and E (gE) that are immune evasion molecules that inhibit antibody and complement. Our vaccine strategy builds upon the immunity provided by gD and blocks the ability of the virus to evade antibody and complement attack. 3) Modify antigen delivery methods. We use nucleoside-modified mRNA, rather than subunit proteins as immunogens. We report preliminary results in mice using a trivalent vaccine that contains modified mRNA encoding glycoproteins C, D and E (gC2/gD2/gE2) administered in lipid nanoparticles. The modified mRNA replaces uridine nucleosides with 1-methylpseudouridine residues. We achieve sterilizing immunity in 63/64 (98%) mice compared with 14/20 (70%) immunized with subunit antigens. We will extend this exciting result to include studies in nave and HSV-1 positive guinea pigs as a more stringent test of vaccine efficacy, evaluate male and female animals, and determine cross-protection against genital HSV-1. 4) Define the immune correlates required to achieve sterilizing immunity. We will assess serum ELISA titers, neutralizing titers, antibodies that block cell-to-cell spread, antibodies that block gC and gE immune evasion, antibody-dependent cellular cytotoxicity titers, and mucosal ELISA and neutralizing antibodies as immune correlates of protection. These studies will help establish immunogenicity targets for future human trials. 5) Determine whether mRNA immunization produces antibodies to crucial epitopes. The crucial epitopes include those on gC and gD that neutralize virus, gC and gE that inhibit complement and antibody, and gD and gE involved in cell-to-cell spread. We will use a novel high throughput biosensor platform to determine whether immunization produces antibodies to these crucial epitopes. Measuring epitope specific antibody responses will identify gaps in immunogenicity and guide us on those epitopes to modify to enhance vaccine protection. The four labs have the combined expertise to develop a genital herpes vaccine that achieves sterilizing immunity in animals and subsequently in humans.